Human amyloid-β enriched extracts: evaluation of in vitro and in vivo internalization and molecular characterization

Abstract

Background Intracerebral inoculation of extracts from post-mortem human Alzheimer’s disease brains into mice produces a prion-like spreading effect of amyloid-β. The differences observed between these extracts and the synthetic peptide, in terms of amyloid-β internalization and seed and cell-to-cell transmission of cytosolic protein aggregates, suggest that brain extracts contain key contributors that enhance the prion-like effect of amyloid-β. Nevertheless, these potential partners are still unknown due to the complexity of whole brain extracts. Methods Herein, we established a method based on sequential detergent solubilization of post-mortem samples of human brains affected by Alzheimer’s disease that strongly enrich amyloid-β aggregates by eliminating 92% of the remaining proteins. Internalization of Aβ 1–42 from the enriched AD extracts was evaluated in vitro, and internalization of fluorescent-labeled AD extracts was also investigated in vivo. Furthermore, we carried out a molecular characterization of the Aβ-enriched fraction using label-free proteomics, studying the distribution of representative components in the amygdala and the olfactory cortex of additional human AD brain samples by immunohistochemistry. Results Aβ 1–42 from the enriched AD extracts are internalized into endothelial cells in vitro after 48 h. Furthermore, accumulation of fluorescent-labeled Aβ-enriched extracts into mouse microglia was observed in vivo after 4 months of intracerebral inoculation. Label-free proteomics (FDR < 0.01) characterization of the amyloid-β-enriched fraction from different post-mortem samples allowed for the identification of more than 130 proteins, several of which were significantly overrepresented (i.e., ANXA5 and HIST1H2BK; p < 0.05) and underrepresented (i.e., COL6A or FN1; p < 0.05) in the samples with Alzheimer’s disease. We were also able to identify proteins exclusively observed in Alzheimer’s disease (i.e., RNF213) or only detected in samples not affected by the disease (i.e., CNTN1) after the enrichment process. Immunohistochemistry against these proteins in additional tissues revealed their particular distribution in the amygdala and the olfactory cortex in relation to the amyloid-β plaque. Conclusions Identification and characterization of the unique features of these extracts, in terms of amyloid-β enrichment, identification of the components, in vitro and in vivo cell internalization, and tissue distribution, constitute the best initial tool to further investigate the seeding and transmissibility proposed in the prion-like hypothesis of Alzheimer’s disease. Electronic supplementary material The online version of this article (10.1186/s13195-019-0513-0) contains supplementary material, which is available to authorized users.

HIV-1 and Amyloid Beta Remodel Proteome of Brain Endothelial Extracellular Vesicles

“…Similar obstacles were described in another proteomic study, in which Aβ was not identified in human AD brains. However, Aβ was detected by dot blot and ELISA from the same samples [29], supporting the notion that the lack of Aβ detection in the proteome was likely due to technical limitations.…”

“…In addition, HIST1H2BK has been one of the unique proteins in the EV total proteome from the Aβ group. In contrast, RNF213 was not identified in any of our EV samples, although it was unique to the AD brain samples and also found within the amyloid plaques [29]. One explanation for this phenomenon could be that RNF213 in the AD brain might not originate from brain endothelial cells.…”

“…Some of the unique proteins identified in our HBMEC-derived EVs exhibit a substantial overlap with proteins detected by label-free proteomics in Aβ-enriched extracts from human AD brains [29], suggesting the relevance of EV proteins to Aβ pathology. The examples include ANXA5, FGB, LAMA5, and VIM found both in the total proteome of EVs in the HIV group and in Aβ-enriched extracts from human AD brains [29]. In addition, specific types of tubulins, such as TUBA1B and TUBB4B, were present, although they did not change in AD brains.…”

“…In addition, specific types of tubulins, such as TUBA1B and TUBB4B, were present, although they did not change in AD brains. Among the unique proteins in the HIV+Aβ group's total proteome, FGG and HIST1H2BK, as well as tubulins TUBB and TUBB2A, were also enriched in extracts from AD brains [29]. In addition, HIST1H2BK has been one of the unique proteins in the EV total proteome from the Aβ group.…”

Integrative Multi-Omics Analysis in Calcific Aortic Valve Disease Reveals a Link to the Formation of Amyloid-Like Deposits

“…Moreover, a complex of TTR, APOA-1 and C3 might be involved in Aβ clearance [ 64 , 65 ]. In addition, HBB and TGFBI were recently identified in Aβ-enriched extracts from Alzheimer’s patients [ 66 ]. Cumulative experimental evidence showed that Aβ interacts with different coagulation factors, promoting a prothrombotic and proinflammatory milieu [ 67 , 68 , 69 ].…”